Regis R. Vollmer

Hydroporation as the mechanism of hydrodynamic delivery.

We have reported that a rapid tail vein injection of a large volume of plasmid DNA solution into a mouse results in high level of transgene expression in the liver. Gene transfer efficiency of this hydrodynamics-based procedure is determined by the combined effect of a large volume and high injection speed. Here, we show that the hydrodynamic injection induces a transient irregularity of heart function, a sharp increase in venous pressure, an enlargement of liver fenestrae, and enhancement of membrane permeability of the hepatocytes. At the cellular level, our results suggest that hepatic delivery by the hydrodynamic injection is accomplished by the generation of membrane pores in the hepatocytes.

Adrenal epinephrine and norepinephrine release to hypoglycemia measured by microdialysis in conscious rats

Experiments were conducted in conscious male rats to determine whether hypoglycemia induced by insulin administration preferentially stimulated epinephrine (Epi) or norepinephrine (NE) adrenal medullary chromaffin cells. The release of Epi and NE from the adrenal medulla was continuously monitored using a microdialysis probe of novel design that had been inserted in the adrenal medulla approximately 16 h before the administration of insulin. Following insulin, 3 U/kg i.v., blood glucose declined and dialysate Epi levels rose. No measurable increment in dialysate NE was obtained. Similarly, plasma Epi increased with no detectable change in NE. Patterns of dialysate and plasma catecholamine changes were similar in two groups of animals that had been fed or fasted overnight before insulin treatment. However, the magnitude of the Epi increase was greater in the fasted animals. After recovery of the blood glucose concentration to preinsulin levels, dialysate and plasma catecholamine concentrations returned to control values. These experiments clearly demonstrate that adrenal medullary chromaffin cells that produce Epi are preferentially stimulated in response to insulin-induced hypoglycemia.Experiments were conducted in conscious male rats to determine whether hypoglycemia induced by insulin administration preferentially stimulated epinephrine (Epi) or norepinephrine (NE) adrenal medullary chromaffin cells. The release of Epi and NE from the adrenal medulla was continuously monitored using a microdialysis probe of novel design that had been inserted in the adrenal medulla ∼16 h before the administration of insulin. Following insulin, 3 U/kg iv, blood glucose declined and dialysate Epi levels rose. No measurable increment in dialysate NE was obtained. Similarly, plasma Epi increased with no detectable change in NE. Patterns of dialysate and plasma catecholamine changes were similar in two groups of animals that had been fed or fasted overnight before insulin treatment. However, the magnitude of the Epi increase was greater in the fasted animals. After recovery of the blood glucose concentration to preinsulin levels, dialysate and plasma catecholamine concentrations returned to control values. These experiments clearly demonstrate that adrenal medullary chromaffin cells that produce Epi are preferentially stimulated in response to insulin-induced hypoglycemia.

Oxytocin null mice ingest enhanced amounts of sweet solutions during light and dark cycles and during repeated shaker stress

Central oxytocin (OT) pathways appear to limit consumption of sweet solutions. Male and female C57BL/6 mice that lack the gene for oxytocin (OT KO mice) displayed an initial and sustained enhanced intake of sucrose solution over water compared to wild type (WT) mice when the solutions were presented as a two-bottle choice [Amico JA, Vollmer RR, Cai HM, Miedlar JA, Rinaman R. Enhanced initial and sustained intake of sucrose solution in mice with an oxytocin gene deletion. Am J Physiol: Regul Integr Comp Physiol 2005;289:R1798-806]. In this study we examined the ingestion of a non-nutritive sweetener, 0.2% saccharin in sucrose-experienced OT KO and WT mice given a two-bottle choice between saccharin solution and water available ad libitum for 4 days. Compared to WT mice, OT KO mice consumed significantly greater volumes of saccharin solution during the dark and light photoperiods on the first day and subsequent days of the study. The results were replicated when the experiment was repeated in the same animals. In another experiment, we determined that daily exposure to platform shaker stress did not alter the marked sucrose consumption in OT KO mice. OT KO mice drank significantly more sucrose than WT mice during periods of stress and non-stress. We conclude that the avid consumption of sweetened solutions by OT KO mice is not restricted to a single photoperiod, occurs independent of caloric content of the sweetened solution, and is not altered by exposure to the daily stress of platform shaker. The cumulative results from our studies of sucrose and saccharin ingestion in OT KO and WT male and female mice suggest a special role for sweet taste in the recruitment of OT neurons.

Selective Neural Regulation of Epinephrine and Norepinephrine Cells in the Adrenal Medulla-Cardiovascular Implications

The innervation of the adrenal medulla regulates the release of catecholamines from the two, epinephrine (EPI) and norepinephrine (NE), populations of chromaffin cells. Adjustments in the neural output to the adrenal medulla are made by centers in the brain that integrate the sensory input arising from a variety of challenges and the resulting changes in secretion assist in the restoration of homeostasis. Interestingly, the adrenal medullary secretory responses do not simply reflect increments a fixed ratio of EPI to NE as might be expected if release was proportional to the number EPI and NE cells. Instead, the ratio of EPI to NE changes depending on the magnitude and type of stimulus that initiates neural activation of the medulla. The variability in the EPI:NE release ratio implies that the EPI and NE cells can be differentially stimulated. Although the underlying mechanisms are not fully characterized, this review presents an emerging view that the selective control of EPI and NE cells is accounted for, first, by the existence of separate neural circuits between brain centers and the chromaffin cells, and second, through neuromodulation that selectively influences EPI and NE cells. The presence of mechanisms that allow for separate control of the EPI and NE cells may significantly augment the range of cardiovascular and metabolic responses mediated through activation of the adrenal medulla.

Centrally administered oxytocin elicits exaggerated grooming in oxytocin null mice

Experiments were conducted to determine if the chronic absence of the neurotransmitter oxytocin (OT) in null mice resulted in alterations in the responsiveness and abundance of central OT receptors. Self-grooming elicited by intracerebroventricularly administered OT was studied as an indicator of the activation of central OT receptors and autoradiography was used to map the distribution and density of OT receptors in OT null and wild type mice. The intracerebroventricular administration of OT, but not vehicle, artificial cerebrospinal fluid (aCSF), produced a robust increase in grooming behavior in both OT null and wild type animals, P<.001. However, OT-induced grooming was significantly greater in OT null than wild type mice, P<.005. The enhanced grooming was selective to OT as indicated by the finding that grooming to intracerebroventricular arginine vasopressin (AVP) was of the same magnitude in both OT null and wild type mice. OT-induced grooming appears to be mediated through the activation of OT receptors because pretreatment of animals with an OT antagonist, Atosiban, abolished OT-induced grooming, but not AVP-induced grooming. OT receptor distribution and binding in brains of OT null and wild type mice were examined by autoradiography and were not significantly different. The results indicate that the chronic absence of OT in null mice leads to an increase in OT receptor responsiveness that contributes to the augmented grooming activity elicited by centrally administered OT.

ADRENAL MEDULLARY CATECHOLAMINE SECRETION PATTERNS IN RATS EVOKED BY REFLEX AND DIRECT NEURAL STIMULATION

Epinephrine (EPI) and norepinephrine (NE), secretion patterns evoked by reflex (to hypotension and hypoglycemia) and direct neural stimulation of the adrenal medulla were measured in pentobarbital anesthetized male Sprague-Dawley rats. Secretion rates were determined by collecting adrenal venous blood. Baseline catecholamine secretion was similar in innervated and denervated glands indicating that there was little tonic sympathetic neural drive to the medulla. Both hydralazine-induced hypotension and insulin-induced hypoglycemia significantly increased catecholamine secretion with the secretion of EPI predominating. Similarly, in response to stimulation of the splanchnic nerve, frequency-related increments in EPI and NE were elicited with EPI release being greater than NE at all frequencies. However, the magnitude of the increase in secretion during splanchnic stimulation at frequencies above 1 hz greatly exceeded the release achieved by reflex stimulation. The results indicate that despite the fact that the stimuli of hypotension and hypoglycemia are integrated by different centers in the brain, the pattern of adrenal release is similar.

Corticosterone release is heightened in food or water deprived oxytocin deficient male mice.

Recent studies in female mice that cannot synthesize oxytocin (OT) suggest that central OT neural pathways attenuate the response of the hypothalamic-pituitary-adrenal (HPA) axis to certain stressors. OT deficient (OT-/-) female mice had higher plasma corticosterone concentrations than wild type (OT+/+) female mice following exposure to platform shaker (Mantella et al., 2004). The present study examined the corticosterone response of OT-/- and OT+/+ male mice that were exposed to shaker stress or other stressors (i.e., administration of cholecystokinin (CCK), dehydration, or fasting) that are known to activate central OT neurons in mice. Plasma corticosterone concentrations were higher in male mice receiving each stress than in male mice not exposed to a stressor. Plasma corticosterone concentrations were higher in OT-/- than OT+/+ male mice that were water deprived (P < 0.05) or fasted (P < 0.03), whereas corticosterone concentrations following exposure to platform shaker or CCK administration (10 microg/kg i.p.) were not different between genotypes. These findings support the hypothesis that absence of OT results in a heightened response of the HPA axis to certain stressors and that OT can attenuate the corticosterone response associated with overnight food or water deprivation in male mice.

Low Sodium Diet Augments Plasma and Tissue Catecholamine Levels in Pithed Rats

Plasma and tissue (cardiac, vascular, renal, and adrenal) catecholamine concentrations were measured in pithed male Wistar rats maintained on low (10 mEq/kg diet), basal (115 mEq/kg diet), or high (1200 mEq/kg diet) sodium test diets for five weeks. Significant differences in catecholamine disposition were observed only in response to sodium restriction; responses to basal and high sodium intakes were consistently similar. Baseline plasma catecholamine levels (p less than 0.01) as well as those in response to stimulation of the entire sympathetic outflow at 4 Hz were markedly enhanced in low sodium rats (p less than 0.001). The facilitation of stimulation-induced increments in plasma norepinephrine levels in low sodium rats may be related to the finding that norepinephrine content was also elevated in noradrenergically innervated tissues (atria, ventricles, mesenteric artery, and kidneys) (p less than 0.01). Adrenal catecholamine levels, however, were not affected by dietary sodium restriction. Despite the peripheral catecholamine changes associated with a low sodium intake, pressor and tachycardic responses to sympathetic nerve stimulation were similar across dietary sodium groups. The results indicate that a low sodium intake enhances plasma and tissue catecholamine levels, adaptations that may be important in the maintenance of sympathetic responsiveness.

Oxytocin knockout mice: a model for studying stress-related and ingestive behaviours.

Oxytocin (OXT) that is released centrally is believed to be anxiolytic and have stress-attenuating effects. Oxytocin knockout (OXTKO) mice, a genetic model of OXT deficiency, have heightened corticosterone release after acute stress and greater anxiety-related behaviour in an elevated plus maze compared to wild-type (WT) mice. In the present set of experiments, we recorded the rise in body temperature, referred to as stress-induced hyperthermia (SIH), following transfer to a metabolic cage, which triggers both anxiety and corticosterone release in mice. SIH is a marker of activation of the hypothalamic-pituitary-adrenal (HPA) axis and sympathetic nervous system. Because corticosterone release after acute stress is typically greater in OXTKO than in WT mice, we measured SIH as a surrogate marker of corticosterone release. Following transfer to a metabolic cage, both OXTKO and WT mice increased body temperature, but to the same degree. Pregnant mice, which are known to have blunted corticosterone release to acute stress, had attenuated SIH after transfer to a metabolic cage compared to cycling mice, but both genotypes manifested the same degree of attenuation. In addition, we tested the effects of the cannabinoid receptor 1 (CBR1) antagonist/inverse agonist (AM251) upon feeding and SIH in OXTKO versus WT mice. CBR1 antagonists are known to diminish food intake and to enhance corticosterone both basally and following acute stress. Although AM251 blunted food intake, the effect was equivalent in both genotypes. The agent did not affect the SIH response compared to mice treated with vehicle. SIH is excellent for defining anxiolytic or blunted corticosterone responses (such as the stress hyporesponsiveness of pregnancy), but is limited in its ability to detect the heightened corticosterone responses that have been reported in OXTKO mice following exposure to psychogenic stress.

Corticosterone release in oxytocin gene deletion mice following exposure to psychogenic versus non-psychogenic stress.

Both anxiety-related behavior [J.A. Amico, R.C. Mantella, R.R. Vollmer, X. Li, Anxiety and stress responses in female oxytocin deficient mice, J. Neuroendocrinol. 16 (2004) 1-6; R.C. Mantella, R.R. Vollmer, X. Li, J.A. Amico, Female oxytocin-deficient mice display enhanced anxiety-related behavior, Endocrinology 144 (2003) 2291-2296] and the release of corticosterone following a psychogenic stress such as exposure to platform shaker was greater in female [J.A. Amico, R.C. Mantella, R.R. Vollmer, X. Li, Anxiety and stress responses in female oxytocin deficient mice, J. Neuroendocrinol. 16 (2004) 1-6; R.C. Mantella, R.R. Vollmer, L. Rinaman, X. Li, J.A. Amico, Enhanced corticosterone concentrations and attenuated Fos expression in the medial amygdala of female oxytocin knockout mice exposed to psychogenic stress, Am. J. Physiol. Regul. Integr. Comp. Physiol. 287 (2004) R1494-R1504], but not male [R.C. Mantella, R.R. Vollmer, J.A. Amico, Corticosterone release is heightened in food or water deprived oxytocin deficient male mice, Brain Res. 1058 (2005) 56-61], oxytocin gene deletion (OTKO) mice compared to wild type (WT) cohorts. In the present study we exposed OTKO and WT female mice to another psychogenic stress, inserting a rectal probe to record body temperature followed by brief confinement in a metabolic cage, and measured plasma corticosterone following the stress. OTKO mice released more corticosterone than WT mice (P<0.03) following exposure to this stress. In contrast, if OTKO and WT female and male mice were administered insulin-induced hypoglycemia, an acute physical stress, corticosterone release was not different between genotypes. The absence of central OT signaling pathways in female mice heightens the neuroendocrine (e.g., corticosterone) response to psychogenic stress, but not to the physical stress of insulin-induced hypoglycemia.